ARF6在糖尿病肾病足细胞胆固醇代谢障碍及损伤中的作用及机制

Podocyte injury or decreased podocyte number (podocytopenia) has been described as an independent predictor of diabetic nephropathy（DN）progression，and increased cholesterol accumulation is associated with podocyte injury，but the mechanism of cholesterol metabolism dysfunction in podocyte under DN condition remains unclear. We have recently found that high glucose inhibit ARF6 expression via gene screening of 25 cholesterol transport related molecules, and cholesterol accumulation and cell injury were observed in high glucose-treated podocyte. This project intends to investigate the role of ARF6 in podocyte cholesterol metabolism dysfunction and injury under DN condition. Firstly, STZ-induced DN model, high glucose-treated podocyte and biopsy specimens from DN patients will be used to evaluate the expression of ARF6, cholesterol metabolism dysfunction and podocyte injury; Secondly, we will explore the role of ARF6 in regulation of podocyte cholesterol metabolism via altering the expression and activity of ARF6 with its mutated plasmids , stable ARF6 knock out podocyte line by CRISPR/Cas9 technology; Finally, we will explore the potential mechanism of ARF6 in cholesterol metabolism dysfunction in high glucose-treated podocyte by detection and expression interference of the downstream molecules. This project will provide a theoretical evidence for ARF6 to be a new therapeutic target of DN.

足细胞损伤/减少是糖尿病肾病（DN）进展的独立预测因素，既往研究发现胞内胆固醇蓄积与DN患者足细胞损伤相关，但DN状态下足细胞胆固醇代谢失衡的机制仍不清楚。本项目组前期通过对既往报道的25个胆固醇转运相关基因筛查，发现ARF6在高糖刺激的足细胞中表达显著下调，且高糖可诱导足细胞内胆固醇蓄积及损伤。本项目拟围绕ARF6在DN足细胞胆固醇代谢障碍与损伤中的作用及机制进行研究。通过对STZ诱导DN大鼠模型、培养足细胞及DN患者肾活检标本的研究，评价ARF6表达与足细胞胆固醇代谢障碍及损伤的关系；通过ARF6活性突变质粒转染、CRISPR/Cas9技术构建ARF6稳定敲除足细胞株，探讨ARF6表达及活性改变对足细胞胆固醇代谢及损伤的影响；通过对下游信号分子的检测及干扰明确ARF6在高糖状态下介导足细胞胆固醇代谢障碍的机制。本研究将为ARF6成为DN治疗的新靶点提供理论依据。

足细胞损伤/减少是糖尿病肾病（DN）进展的独立预测因素，既往研究发现胞内胆固醇蓄积与DN患者足细胞损伤相关，但DN状态下足细胞胆固醇代谢失衡的机制仍不清楚。本项目旨在探讨ARF6在糖尿病肾病足细胞胆固醇代谢障碍及损伤中的作用及机制。本研究发现链脲佐菌素诱导糖尿病肾病大鼠模型及高糖处理体外培养足细胞都可导致足细胞胞内胆固醇蓄积及足细胞损伤，且胆固醇蓄积与足细胞ARF6表达下降相关。干扰ARF6表达可进一步加重高糖诱导的足细胞内胆固醇蓄积，诱导足细胞凋亡。正常情况下，干扰ARF6表达，可模拟高糖对足细胞ABCA1表达的效应，诱导ABCA1移位至溶酶体中降解，表达下降，胆固醇外流障碍；高糖刺激下，干扰ARF6表达可进一步加重高糖诱导的足细胞ABCA1降解，胆固醇外流进一步抑制，蓄积增加，损伤加重。 结论：ARF6通过调控胆固醇外流关键分子ABCA1的降解参与足细胞胆固醇代谢的调节；高糖等病理状态下足细胞ARF6表达下调，促使ABCA1降解增加，进而抑制足细胞胞内胆固醇外流，致使胞内胆固醇蓄积及细胞损伤。